All our work is done on frozen sections, snap frozen tissue, and fixed with
acetone or acetone alcohol, we never use paraformaldehyde (PFA) since PFA
causes tends to cause autofluorescence that clashes with FITC) besides
having the additional problem of retrieval, we never have to do that.  

Two example of how we do this with success:

All fluorescent labeled antibodies are incubated in dark to prevent
photobleaching. 

Normal serum block is a combination of two hosts' serums of your two
different secondaries. The initial blocking can be done here. 
Mouse anti A Primary diluted in 1% goat serum/1% donkey serum (a cocktail
makes sure you can use this diluent for both primaries, you can use BSA for
blocking and in rinse buffers if you wish - molecular biology high quality) 
rinse
Goat antiMouse F(ab')frag of IgG, adsorbed to human - FITC,  diluted in 1 -
5% goat/5% donkey serum
rinse
1 - 5% Mouse serum block for 15 minutes (this binds any residual secondary
you may not have removed from staining)
rinse well
Mouse anti B primary, diluted in 1% donkey serum
rinse
Donkey anti-mouse (F(ab')2 frag of IgG-Rhodamine, adsorbed to human diluted
in 1% goat/1% donkey serum
Rinse well.
Use high quality aqueous antifade mounting media.  Examine and take
pictures immediately.  

Caveat:  Do not use two secondaries that are from the same host, these must
be different, you can use goat and rabbit, goat and donkey, donkey and
rabbit; swine and goat, or rabbit, or donkey. Be careful how you set this up. 

Our favorite method is to use one biotinylated primary and one purified
primary antibody.  

Normal serum blocking:  5% goat serum
If your tissue contains endogenous biotin, you must do an avidin biotin
block. 
Biotinylated primary A, the negative control is biotin conjugated mouse IgG
from Jackson or an isotype matched IgG - biotinylated.  
Rinse well
Strepavidin-Alexa 488 (Molecular probes) an equivalent to FITC, does not
photobleach rapidly and extremely bright. SA-FITC is available. 
Rinse well 
1 -5% mouse serum block for 15 minutes
Rinse well
Mouse antihuman B primary
rinse well
Goat or donkey antihuman F(ab')frag of IgG Rhodamine, adsorbed to human
rinse well.  There is a new rhodamine (RRX) flourochrome from Jackson
Immunoresearch that is brighter, resists photobleaching.  Check this out on
Jackson website, their discussion of fluroescent markers is excellent,
informative - worth reading.  
Antifade mounting media, aqueous, examine immediately. 

F(ab')2 frag of IgG eliminate the fc receptor cross reactivity of secondary
to fc receptors on tissue.

It is important to do the protocol in sequence with two primaries from the
same host, you do not want cross reactivity. 

You might have success with a directly conjugated primary (FITC) then mouse
serum block, followed by purified primary/secondary labeled with TRITC.  

We can examine these stained sections with either epifluorescent microscope
OR confocal laser scanning microscope. 





Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu